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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Antioxidant Status of Pair-Fed Labrador Retrievers Is Affected by Diet Restriction and Aging^1,2

Howard D. Stowe^*,3,4, Dennis F. Lawler and Richard D. Kealy

^* Professor Emeritus, Michigan State University, Owosso, MI 48867 and Nestle Purina Petcare Company, St. Louis, MO 63164

⁴ To whom correspondence should be addressed. Email: hdstowe{at}msu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Twenty-four sibling pairs of 8-wk-old Labrador Retrievers were assigned to an experiment to determine the effects of diet restriction (75% of control-fed pair mate) on the quality and span of canine life and to identify biological markers of aging in dogs. The antioxidant status of these dogs was monitored by annual assays for serum retinol (RT), retinyl palmitate (RP), total vitamin A (VA), vitamin E (VE), selenium (Se), copper (Cu), and ceruloplasmin (Cp), plasma ascorbic acid (AA), uric acid (UA), and total peroxyl-radical trapping activity (TRAP), and whole-blood glutathione peroxidase (Gpx). Data in this report are for the 6-y period of the experiment when the dogs were between 5 and 10 y of age. Diet restriction reduced RT, VE, Cu, and Cp. Aging was associated with decreased RP, VA, VE, Se, and Cu and with increased RT, Cp, and Gpx. Female dogs had lower RP, VA, Cu, and Cp than male dogs. Litter effects were observed for VE, Cu, UA, and Gpx. Treatment effects on serum RT and Cu suggest that these variables are not as regulated homeostatically by hepatic storage as in most other species. Although the antioxidant profiles did not elucidate how diet restriction contributes to longevity, they have the potential to enhance our understanding of canine clinical nutrition and to have practical applications in formulating canine diets.

KEY WORDS: • diet restriction • dogs • antioxidants • aging

Understanding the processes of aging, with the hope of extending the span of a quality life, is a common goal in human and veterinary medicine. In the case of veterinarians, and particularly those specializing in companion animals, an important component of this objective is to extend the duration of the human/animal bonds that are an important aspect of pet ownership. Interest in ways to modulate the aging processes of humans and animals was given momentum between 1935 and 1939 by reports that the life spans of rodents (mice and rats) could be markedly increased by restricted energy intake (40% of ad libitum consumption) without malnutrition (1,2). The very earliest observations on diet-restricted rodents, however, are attributed to Moreschi (3) and Rouse (4), who demonstrated how limited feeding delayed the growth of tumor transplants in some strains of mice.

During the intervening years, diet restriction was shown to increase the life span of other species including fish, spiders, rotifers, and fleas (5). At least 2 studies are ongoing to determine whether the life extension associated with diet restriction can be achieved in nonhuman primates (6,7).

Despite many studies, the biochemical and physiological processes associated with the role of diet restriction in extending life span have not been established conclusively. One of the major, although controversial, hypotheses is that diet restriction slows the metabolic rate, which in turn reduces endogenous processes by which DNA and other macromolecules can be damaged during metabolism (8).

The damage is produced during normal aerobic metabolism by reactive endogenous by-products and should, therefore, be spared by appropriate concentrations of in vivo antioxidants (9). In the absence of data on the effects of diet restriction in dogs, a long-term experiment was established in 1987 to provide information on this topic (10). This report summarizes the antioxidant data for 6 y (dog ages 5–10 y) of the experiment and provides clinical reference values, many previously unavailable, for antioxidants in canine blood, serum, and plasma.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Dogs and housing. Labrador Retrievers (n = 48) from 7 litters from the Nestle Purina breeding colony at Gray Summit, MO were assigned to a paired-feeding experiment starting at 8 wk of age after acclimation to co-penning and individual feeding stalls (10). Each of the 24 pairs of consisted of same-gender full siblings fed the growth formula (Table 1) from 8 wk to 3.5 y of age. During this period, the growth formula was consumed ad libitum by the control dogs, whereas their paired mates were restricted to 75% of the ad libitum intake. Thereafter, and to the end of their lives, the adult formula (Table 1) was fed at a rate that provided 260 kJ of digestible energy/(kg body weight·d) for the control-fed (CF)⁵ dogs and 75% of that amount [195 kJ DE/(kg body weight·d)] for the diet-restricted (DR) dogs. The micronutrient concentrations of the adult formula are presented in Table 2.

    Assay methods. Vitamin A [retinol (RT) and retinyl palmitate (RP)] was measured using a HPLC procedure (11). This is an isocratic method using hexane:chloroform (60:40) as the mobile phase pumped at 2.5 mL/min through a microporasil column (Waters Millipore) at 1500 g/cm². Detection was fluorometric at excitation and emission wavelengths of 330 and 470 nm, respectively. Vitamin E (-tocopherol and -tocopheryl acetate) was measured using a modification of the above HPLC procedure (11) wherein the mobile phase was a mixture of hexane and chloroform (85:15) and detection was by UV light at 292 nm.

Selenium was assayed using a phosphoric/nitric acid digestion procedure followed by fluorometric detection (12,13). Copper was measured with an inductively coupled plasma-atomic emission spectrophotometer (Model 955 Atom Comp, Jarrell Ash) at emission line 324.754. Ceruloplasmin was measured by a method in which o-diansidine dihydrochloride is used and absorbance is measured at 540 nm (14).

Ascorbic acid and UA were measured simultaneously by HPLC and electrochemical detection (15). In this assay, the HPLC unit, including a 3.9 x 150 mm C₁₈ Nova-Pak 60 A 4-µm column and a Nova-Pak C₁₈ guard pack precolumn (Waters Millipore), was coupled with a Model 5200 multi-electrode electrochemical detector (Coulochem II, ESA).

The TRAP assays were conducted by a method (16) that uses a Clark-type oxygen electrode (Yellow Springs Instrument) to measure oxygen uptake in samples subjected to controlled peroxidation induced by the addition of 2,2'-azo-bis(2-amidinopropane hydrochloride) (Polysciences).

Glutathione peroxidase was measured via the method of Paglia and Valentine (17) as modified by Anderson et al. (18). Double-strength Drabkin's solution was used as the hemolyzing agent. Gpx concentrations are expressed as enzyme units (EU) per unit of blood and per gram of hemoglobin (Hb); therefore, Hb assays were conducted on each hemolysate using a Model 54 hemophotometer (Fisher Scientific Products).

    Statistical analyses. Within each collection year, data from both dogs in a pair had to be available to be included in the analyses. Data from 2 dogs administered thyroid supplementation were excluded from all years of analysis, and data from 1 diabetic dog were excluded in y 10. Statistical analyses were performed by the General Linear Models procedure of the SAS package (19). Each year, ANOVA, accounting for litter, gender, and pairing within litter and gender, was used to test for treatment differences. ANOVA for repeated measures was used to test for differences over all years. Differences were considered significant when P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Treatment effects. On an individual year basis (Table 3), DR reduced (P < 0.01) serum RT in y 5 and 6, RP in y 5, and VE in y 6 and 7; reduced serum Cu (P < 0.05) in y 6; and reduced plasma TRAP (P < 0.01) in y 6. In the combined 5- to 10-y data (Table 3), DR decreased serum RT (P < 0.05), VE (P < 0.05), Cu (P < 0.01), and Cp (P < 0.01).

    Gender effects. Females (Table 3) had lower serum RP (P < 0.05), total VA (P < 0.01), Cu (P < 0.01), and Cp (P < 0.05) than males.

    Litter effects. Litter effects (Table 3) were found for serum VE (P < 0.01) and Cu (P < 0.05), for plasma UA (P < 0.01), and for Gpx (P < 0.01) per unit of blood and Hb.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Vitamin A. In most species, RT (vitamin A alcohol) is the major form of VA in blood serum/plasma. Concentrations of circulating VA in most species are generally believed to be homeostatically maintained by liver stores until these are depleted by an extended dietary deficiency.

The diet fed to these 48 Labrador retrievers contained (Table 2) 8.96 mg RP (esterified VA)/kg. This VA concentration is well above the estimated minimum VA requirement of 2.048 mg (as RP)/kg diet for growing dogs, even without considering the potential VA derived from carotenoids in the natural feed ingredients (20).

The yearly mean serum RT for the CF and DR dogs over the 6-y observation period (2.7 µmol/L) approximates a published value of 2.24 ± 0.13 µmol/L, representing 37 dogs (21).

Significantly lower serum RT in the DR dogs is consistent with observations in DR rats (22,23). The lower serum RT in DR dogs compared with CF dogs suggests that serum RT might be influenced more by total daily VA intake than by the homeostatic influence of liver storage, although food intake per unit of body weight was the same between the 2 groups. Differences between feeding groups with respect to lean and fat body mass (24) probably also influence serum levels of lipid-soluble vitamins. When hepatic VA content was measured in DR and CF rats for up to 27 mo, hepatic VA (RT, retinyl ester, and total retinoid) was significantly increased by DR as well as by age (25).

Retinyl palmitate data indicate that esterified VA, not RT, is the predominant circulating form of VA in dogs. This phenomenon was reported to occur in most species in the order Carnivora and specifically in Canis lupus familiaris, where the predominant serum VA ester was retinyl stearate followed by RP and retinyl oleate (26).

    Vitamin E. Serum VE is derived from tocopherols present in natural dietary lipids, as well as from exogenous tocopherols added to the diet. The recommended dietary concentration of VE for growing dogs is 22 IU (15 µg tocopherol)/kg (20). The diet used contained 15–16 µg/kg of added VE, as tocopheryl acetate, and had an assayed VE concentration of 28.9 µg/kg.

The range of observed serum VE ( tocopherol) values is in agreement with previous reports on canine serum VE (27–30) but well below that in one report (31). The consistently lower VE values in DR dogs than in CF dogs could be interpreted as evidence that serum VE concentrations reflect VE intake and are a reliable measure of VE status in Labrador Retrievers. However, as previously mentioned, the differences in body lean and fat mass between the 2 groups also may influence differences in serum VE. Serum tocopherol observations in other DR species are inconspicuous, but tocopherol concentrations were reduced in muscle tissue in DR compared with CF swine (32).

The decline in VE with age (Table 3) for both DR and CF dogs, until y 8, was steady and approached 50% in magnitude. This finding prompts questions about the optimal dietary VE concentration for aging dogs, and about attempting to limit this decline by increasing dietary VE concentrations in geriatric canine diets. However, the fact that no VE deficiency signs occurred probably indicates that the change is a normal part of the aging process.

The litter effect on VE is possible evidence of a genetic influence on VE utilization. It therefore should be possible to manipulate serum VE concentrations through selective breeding, but the practical relevance of this possibility is probably minor.

    Selenium. Se is an essential trace element that can be toxic in relatively low dietary concentrations. The current FDA-allowable Se supplementation rate for the major food and fiber producing species is 0.3 mg/kg dry diet (33). Although not specifically included in this legislation for livestock, moderate Se supplementation of canine diets is industry practice. The assayed Se concentration of the adult formula (Table 2) in this experiment was 0.21 mg/kg dry weight.

The range of serum Se in the Labrador Retrievers (2.67–3.10 µmol/L) is higher than for many other species (34). The practical relevance of the decline in serum Se in dogs between 5 and 10 y of age seems inconsequential because of their apparent ability to maintain relatively high serum Se concentrations with a modest Se intake.

    Glutathione peroxidase. Gpx is a Se-dependent enzyme that has antioxidant properties and is incorporated into erythrocytes only at the time they are formed in the bone marrow or other hematopoietic centers. Canine Gpx values are comparable to those of other species that have relatively high serum Se values, such as mature sows (34). No reference values for Gpx in dogs were found.

Because the Gpx measured is a Se-dependent enzyme, a strong positive correlation between serum Se and blood Gpx exists in many species (35). For this reason, Se-dependent Gpx assays are used as an indirect measure of Se status in farm livestock. In the Labrador Retrievers, however, the correlation between blood Gpx and serum Se over time was positive only during 4 of the 6 y, and was significant (P < 0.05) only during y 9. Thus, it appears that blood Gpx may not be a reliable indirect assay of Se status in domestic dogs.

The increase in Gpx with age in the dogs is in contrast to the decline in Gpx with age observed in rats (36). The litter effects on Gpx in dogs may be comparable to the reported genetic influence on the Se status of swine (37).

    Ascorbic acid. Dogs are presumed to meet their AA requirement through endogenous synthesis from glucuronic acid; therefore, they do not require exogenous AA. Nevertheless, supplemental AA has been recommended as an alternative treatment for chronic movement disorders in dogs (38).

The AA concentration of the adult dog diet of < 40 mg/kg (Table 2) resulted in yearly mean plasma AA values ranging from 27.5 to 49.5 µmol/L. A mean plasma concentration of 22.7 µmol/L was reported from 1-time assays of 25 dogs (27), which is below any of the yearly mean AA concentration of the Labrador Retrievers. Although plasma AA was unaffected by DR, age, gender, or litter in this study, plasma AA was significantly lowered by both DR and aging in mice (39).

    Uric acid. Circulating UA is considered a major in vivo antioxidant, at least in humans, a species that circulates relatively large amounts of UA in plasma (200–400 µmol/L) (40) compared with a reported reference range of 6–120 µmol/L for UA in canine serum (41). That range encompasses the range of yearly mean plasma UA concentrations (Table 3) of 10–35 µmol/L for the Labrador Retrievers.

There was a tendency (P < 0.10) for plasma UA, for y 7, 8, and 9, to be reduced by DR, suggesting that this variable may be affected by some dietary component(s) or configuration. For instance, egg-based diets significantly affect UA metabolism in dogs (42) but the adult formula did not contain an egg component. The litter effect on UA may be of genetic interest.

    Copper. Cu is an essential trace element because of its requirement for the in vivo formation of several enzymes [lysyl oxidase, ferroxidase (Cp); cytochrome oxidase, tyrosinase, and superoxide oxidase] important in health maintenance.

The minimal recommended dietary Cu concentration for dogs is 2.9 mg/kg (20). The assayed Cu concentration of the adult formula (Table 2) of 12.8 mg/kg on a dry basis resulted in an overall mean serum Cu concentration of 10.9 ± 0.47 µmol/L in the Labrador Retrievers. This compares favorably with a previously reported serum Cu concentration of 611 µg/L (9.62 µmol/L) in Cu-adequate dogs (43).

The reduction of serum Cu values in the DR dogs was unexpected given the Cu content of their diet. In other species, serum Cu concentrations are believed to be homeostatically controlled by hepatic Cu stores until the latter become diminished by prolonged dietary inadequacy. Because the diet of the Labrador Retrievers should not have diminished hepatic Cu stores, Cu homeostasis in this breed may not be regulated by hepatic Cu reserves to the degree anticipated. A reduction in serum Cu in DR Suffolk and Rambouilet ewes was reported (44).

The gender effect on serum Cu was unexpected and was not previously reported. The dog data suggest that, under comparable environments, female dogs may be expected to have lower serum Cu concentrations than male dogs. However, this issue is clouded by the fact that many of the females were ovariohysterectomized during the latter half of the study, and the influence of this on serum Cu is not known.

    Ceruloplasmin. Cp is formed in the liver from newly absorbed Cu and contains 65% of the Cu in serum. Perhaps due to a peculiar amino acid configuration in canine albumin, the Cp content of dog serum is relatively low compared with other species (45). The decrease in serum Cp due to DR is consistent with the treatment effect on serum Cu. Also, the gender effect on Cp was consistent with the gender effect on serum Cu.

    Total peroxyl-radical trapping activity. The TRAP assay is a quantitative measurement of the total antioxidant capability of blood plasma (16). There are no previously published TRAP values for dogs. The range of yearly mean TRAP values for the Labrador Retrievers (156–238 µmol/L) is 25% of the mean TRAP value of 820 ± 148 µmol/L reported for humans.(40) The fact that TRAP values in dogs were unaffected by treatment, age, gender, or litter suggests that various homeostatic mechanisms were successful in maintaining an adequate antioxidant pool in the dogs during the experiment. No references to TRAP assays in other DR species were found.

This long-term experiment provided a unique opportunity to study many aspects of the aging process in dogs and to generate some heretofore unavailable reference values for specific antioxidants in canine blood. The experiment is better appreciated by knowing that even after 10 y, 80% of the original 48 dogs remained on experiment and that by the end of the experiment, the median lifespan of the DR dogs was 13.0 y vs. 11.2 y for the CF dogs, (P < 0.01).

Of the antioxidants studied, only serum RT, VE, Cu, and Cp were influenced by DR. Because DR reduced each of these variables, it seems unlikely that any one of the DR effects would contribute independently to the longevity associated with DR. On the other hand, the age-related changes that occurred, particularly the reductions in VA and VE, could lead to an improved understanding of nutrient metabolism requirements of geriatric canine populations.

	FOOTNOTES

 
¹ Presented at The Purina Pet Institute Symposium: Advancing Life through Diet Restriction; Sept. 20–21, 2002; Hyatt Regency Union Station, St. Louis, MO [Stowe HD, Kealy RD, Lawler DF. Influences of limit-feeding and aging on antioxidant status of pair-fed Labrador Retrievers (abstract); Wilmington, DE: The Gloyd Group; 2002. p. 14].

² Financed by Nestle (formerly Ralston) Purina Company, Checkerboard Square, St. Louis, MO.

³ Steering Committee Member for the diet restriction study and research consultant for its antioxidant segment.

⁵ Abbreviations used: AA, ascorbic acid; CF, control-fed; Cp, ceruloplasmin; DR, diet-restricted; EU, enzyme units; Gpx, glutathione peroxidase; Hb, hemoglobin; RP, retinyl palmitate; RT, retinol; TRAP, total peroxyl trapping activity; U, unit of enzyme activity; UA, uric acid; VA, vitamin A; VE, vitamin E.

Manuscript received 9 February 2006. Initial review completed 22 February 2006. Revision accepted 30 April 2006.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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